Sequence detection system software v2

Using the ISOGEN reagent (Nippon Gene, Tokyo, Japan), total RNAs were extracted from days 11, 15, 17, and 19 deciduas and placentas (n = 3 each day) according to the manufacturer’s protocol. For quantitative real-time PCR (qPCR) analyses, isolated RNA (total 250 ng) was reverse-transcribed to cDNA using ReverTra Ace qRNA RT Kit (Toyobo, Osaka, Japan), and the resulting cDNA (RT template) was stored at 4°C until use [21 (link)].
Reverse-transcribed cDNA was subjected to qPCR amplification using Thunderbird SYBR qPCR Mix Kit (Toyobo) with 0.3 μM of the oligonucleotide primers listed in S1 Table, and qPCR amplification was carried out on an Applied Biosystems STEP One Plus real-time PCR System (Applied Biosystems) [22 (link)]. Amplification efficiencies of each target gene and two reference genes, actin-beta (Actb) and glyceraldehyde 3-phosphate dehydrogenase (Gapdh), were examined through their calibration curves and found to be comparable. The thermal profile for qPCR consisted of 40 cycles at 95°C for 10 sec and annealing and extension at 60°C for 45 sec. Average threshold (Ct) values for each target were determined by Sequence Detection System software v2.2 (Applied Biosystems). Each run was completed with melting curve analysis to confirm the specificity of amplification and the absence of primer dimer.

Using ISOGEN reagent (Nippon gene), total RNAs were extracted from cultured EECs treated with IFNTs, which were performed three times independently. For real-time PCR analyses, isolated RNA (total 0.5 μg) was reverse transcribed to cDNA using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. The cDNA reaction mixture was subjected to real-time PCR amplification using the Thunderbird SYBR qPCR Mix Kit (Toyobo) with primers listed in S1 Table, and PCR amplification was carried out on a Step One Plus real-time PCR System (Applied Biosystems, Foster City, CA). Amplification efficiencies of each target and the reference gene, bovine glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were examined through their calibration curves and found to be comparable. The thermal profile for qPCR consisted of 40 cycles at 95°C for 15 sec, and annealing and extension at 60°C for 60 sec. Average threshold (Ct) values for each target were determined by Sequence Detection System software v2.2 (Applied Biosystems). Each run was completed with a melting curve analysis to confirm the specificity of amplification and the absence of primer dimer [11 (link)].

Using the ISOGEN reagent (Nippon Gene, Tokyo, Japan), total RNAs were extracted from days 15 and 17 conceptuses and endometria and cultured EECs according to the manufacturer’s protocol. For quantitative real-time PCR (qPCR) analyses, isolated RNA (total 250 ng) was reverse-transcribed to cDNA using ReverTra Ace qRNA RT Kit (Toyobo, Osaka, Japan), and the resulting cDNA (RT template) was stored at 4°C until use [21 (link)].
Reverse-transcribed cDNA was subjected to qPCR amplification using Thunderbird SYBR qPCR Mix Kit (Toyobo) with 0.3 μM of the oligonucleotide primers listed in S1 Table, and qPCR amplification was carried out on an Applied Biosystems STEP One Plus real-time PCR System (Applied Biosystems, Foster City, CA) [22 (link)]. Amplification efficiencies of each target gene and two reference genes, bovine beta-action (ACTB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were examined through their calibration curves and found to be comparable [14 (link)]. The thermal profile for qPCR consisted of 40 cycles at 95°C for 10 sec and annealing and extension at 60°C for 45 sec. Average threshold (Ct) values for each target were determined by Sequence Detection System software v2.2 (Applied Biosystems). Each run was completed with melting curve analysis to confirm the specificity of amplification and the absence of primer dimer.